Large-Scale Transfection of Mammalian Cells

The large-scale transfection of mammalian cells allows moderate (milligram to gram) amounts of recombinant proteins (r-proteins) to be obtained for fundamental or clinical research. In this article, we describe a one-liter transfection using polyethyleneimine (PEI) for DNA delivery into human embryonic kidney (HEK-293) cells cultivated in serum-free suspension to produce a recombinant human monoclonal antibody that yields up to about 1 g/L in a 10-day process. The method is based on a DNA delivery step performed at high cell density (20×10(6) cells/mL) by direct addition of DNA and PEI to the culture. Subsequently, the cells are diluted 20-fold for the 10-day production phase in the presence of valproic acid (VPA), a histone deacetylase inhibitor. The methods for plasmid purification, antibody quantification by enzyme-linked immunosorbent assay (ELISA), and affinity purification with protein A are also described.

Development of high-titer transient gene expression processes for manufacturing recombinant proteins

Gaurav Backliwal

The market for recombinant therapeutic proteins (including antibodies) is estimated to be greater than $50-billion and has a compounded annual growth rate of around 20%. More than 50% of all approved processes for manufacturing recombinant proteins use mammalian cells as expression hosts due to their ability to carry out complex assembly and processing, human-like glycosylation and secretion of proteins. This percentage is not expected to change and should even increase with time. Classically, these manufacturing processes use stable cell lines for protein expression, wherein the plasmid coding for the protein of interest is stably integrated into the host cell chromosomes (Stable Gene Expression, SGE). An alternative method for expressing recombinant proteins is Transient Gene Expression (TGE). Herein, the expression of the protein of interest takes place from plasmids which are transfected into the cells and are maintained extra-chromosomally. TGE has become a rapid and easy method for obtaining proteins for structural, biochemical or pre-clinical studies, where only small amounts of one or more proteins might be required. As yet, TGE has not been used for manufacturing recombinant proteins for clinical testing or approved clinical use due to the requirement of large amounts of protein of consistent quality. Even though TGE has been scaled-up to the 100-liter scale and is being attempted at the 1000-liter scale, due to low specific productivity, low volumetric yields and the high cost of DNA, TGE processes are not able to economically produce sufficient amounts of proteins for such purposes. Therefore, the goal of this thesis was to improve TGE in order to create high-titer processes, which would be economically more feasible for manufacturing large-amounts of recombinant proteins. This was achieved by: Improving and simplifying gene delivery – by the combination of high cell density at time of transfection and in-situ complex formation (as apposed to a-priori complex formation), we could enhance protein expression levels and gene delivery. This made implementation more robust and easy, with greater choice of media which could be used and cell density which could be maintained. Improving gene expression – by using a combination of inhibitors of histone deacetylases like valproic acid, and growth factors like acidic fibroblast growth factor, we could enhance specific productivity by ∼20-fold. Enhancing the cell densities at which cells could be maintained after transfection – by the combination of cell cycle regulators like human p18 and p21 and treatment with inhibitors of histone deacetylases, we blocked cell growth which allowed cells to be maintained at significantly higher cell densities, allowing us to enhance volumetric productivity by ∼100-fold. Overall these improvements allowed antibody titers in excess of 1 g/l. The gram per liter range of volumetric productivity has previously only been reported for optimized SGE based manufacturing processes. We therefore improved the economic feasibility of TGE for manufacturing recombinant proteins and reduced the difference in manufacturing costs between TGE and SGE, for a given amount of protein, by an estimated 50-fold. With some further development work and study of related issues like protein quality, TGE based processes can be expected to become part of mainstream recombinant protein manufacturing in the future.

EPFL2008

Transient recombinant protein expression in mammalian cells

Sarah Wulhfard

Transient gene expression (TGE) is a rapid method for generating recombinant proteins in mammalian cells, but the volumetric productivities for secreted proteins in transiently transfected CHO DG44 cells are typically more than an order of magnitude lower than the yields achieved with recombinant CHO-derived cell lines. The goals of the thesis are to identify the limitations to higher TGE yields in CHO DG44 cells and to find possible solutions to overcome the problems. Initially an attempt was made to enhance TGE production by increasing the amount of transfected plasmid DNA. However, this approach did not result in increased recombinant protein levels; on the contrary, transfection with an excess of plasmid DNA (> 1.25 μg/ml) had a negative impact on transgene mRNA levels and protein production. Moreover, it was also observed that recombinant protein yield was strongly dependent on the mRNA level. Therefore, three strategies aimed at increasing the amount of transgene mRNA were investigated. For the first approach, transfected cells were exposed to hypothermic conditions during the production phase. It was already known that lower temperatures increase protein production several fold in recombinant CHO DG44-derived cell lines. The second strategy involved the treatment of transfected cells with valproic acid, a histone deacetylase inhibitor that reduces the effects of gene silencing. The third approach aimed to increase transgene mRNA levels by overexpressing transcription factors and growth factors. With the first two strategies recombinant antibody yields of 60-80 mg/L were achieved whereas the untreated control transfections produced only 5-10 mg/L. Combination of the two strategies led to the production of 90 mg/L of antibody. Moreover, in the treated cultures, the steady-state level of transgene mRNA was 3-5 times higher than in the untreated cultures and remained stable up to 6 days post-transfection. Using the third approach, the increase in recombinant protein production was moderate and transgene mRNA amounts were only 2-fold higher in treated samples compared to the control. When specific proteins such as c-fos, c-jun, NF-kB, and acidic fibroblast growth factor (aFGF) were overexpressed the recombinant antibody production was 20 mg/L compared to 5 mg/L for the control transfection. Overexpression of either a transcription factor or a growth factor in combination with treatment with valproic acid allowed the recombinant protein yield to reach 90 mg/L. However, the benefit of the overexpressed factors was minimal compared to the effect of valproic acid alone. In conclusion, it was demonstrated that the level and stability of transgene mRNA are important factors for increasing volumetric yields in transiently transfected CHO DG44 cells. Furthermore, three approaches aimed to increase mRNA amounts were tested. Exposure to hypothermic conditions and treatment with valproic acid were the two best strategies tested. Both are simple, cost-effective, and scalable making transient gene expression in CHO DG44 cells a feasible alternative for rapid production of gram amounts of recombinant protein.

EPFL2009

The Use of Valproic Acid in Transient Gene Expression with HEK-293E Cells

Divor Kiseljak

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins. Recently, valproic acid (VPA), a well known histone deacetylase inhibitor, has been suggested for the use in cell cultures to enhance recombinant protein expression. However, little was known about protein expression in HEK-293E cells under VPA treatment. Therefore, the first aim of this thesis was to describe the effects of VPA on protein expression by TGE in HEK-293E cells. The addition of VPA induced a dose-dependent increase in protein production. Under the optimal VPA concentration (3.75 mM) IgG volumetric productivity increased over 8-fold compared to the control. This was achieved through improvements in both integral viable cell density (IVCD) and cell specific productivity. The IVCD was improved by the VPA-induced cell growth arrest resulting in longer culture duration and extended protein production phase. The enhancement in specific productivity in VPA-treated cells was a consequence of higher cellular pDNA copy number and improved pDNA utilization that led to higher transgene mRNA levels. The study on limitations governing VPA-based TGE protein expression in HEK-293E cells revealed the bottlenecks at the transcriptional and translational levels, however the limitation did not appear to be at the level of protein folding or assembly for the model IgG antibody studied. In addition, the effect of VPA was tested on the expression of different recombinant monoclonal antibodies and Fc-fusion proteins. The results showed that VPA addition did not always significantly improve protein titers. Since the optimal VPA concentration was dependent on both the recombinant protein being expressed and the amount of coding pDNA used for transfection, we recommend evaluating the suitability of VPA addition and the optimization of VPA concentration in order to find the optimal conditions for TGE protein expression. We believe that this work will contribute to the design of more rational, robust, and successful expression platforms for recombinant protein production in HEK-293E cells. One of the major contributors to the cost of TGE process at large scale is the considerable amount of pDNA required for transfection. Therefore, the second aim of III this thesis was to determine a cost-effective method to reduce the pDNA amount. We showed that a part of the coding pDNA can be replaced with nonspecific (filler) DNA with minimal loss in volumetric productivity. The studies revealed that the presence of filler DNA allowed the coding pDNA to be distributed over a larger number of DNA-PEI complexes. This resulted in a higher percentage of transfected cells and may favor the better utilization of the coding pDNA by the cell’s transcription machinery. The use of filler DNA as a part of defined fed-batch process (based on the addition of glucose and plant derived protein hydrolysates) resulted in 25 fold reduction in coding pDNA amount requirement while maintaining volumetric protein productivity over 1 g/l. The cost reduction of TGE-produced proteins, due to significant decrease in pDNA amount requirement for transfections, will certainly facilitate the development and implementation of large scale TGE as a feasible method for TGE-based protein production.

EPFL2013